Analysis of osteoclast-specificmembrane-bound proteins that are expressed during osteoclast differentiation

破骨细胞分化过程中有破骨细胞特异性功能蛋白的表达。基于这一假设，我们试图鉴定在我们实验室已经建立的破骨细胞特异性抗体（Mab-5H）的抗原（5h抗原）。利用兔未分离骨细胞培养的破骨细胞形成实验系统，我们发现5h抗原在破骨细胞前体中与破骨细胞分化的标记酶酒石酸抗性酸性磷酸酶（TRAP）的表达并行。与甲状旁腺激素（PTH）同时加入Mab-5H后，PTH诱导的破骨细胞的形成和骨吸收活性均被Mab-5H所破坏。这些结果表明，破骨细胞前体特异性产生的5h -氮参与了破骨细胞的分化和功能。5h抗原在破骨细胞中有两种不同的形式，一种是胞质形式，另一种是质膜结合形式。因此，我们试图净化这两种不同的形式。然而，由于我们可以从骨骼中获得的破骨细胞数量很少，因此很难从破骨细胞提取物中纯化抗原。然后，我们寻找除破骨细胞外的其他来源来纯化5h抗原，发现肠道也表达了足够的5h抗原来纯化它们。经SDS-PAGE分析，纯化的5h抗原是一个高度糖基化的蛋白，分子量为65 kDa，非常不稳定。胞质抗原也是一种糖蛋白，在中性条件下分子量为600 kDa，在SDS-PAGE中分子量为65 kDa，更倾向于膜结合形式。然后我们寻找胞质5h抗原的氨基酸序列。然而，由于抗原的n端被阻断，氨基酸分析没有成功。我们也试图分析有限水解产生的抗原肽片段的氨基酸序列，但我们无法获得足够数量的片段进行分析。在本研究中，我们发现5h抗原在破骨细胞前体中主要表达，在破骨细胞的分化和功能中发挥重要作用。然而，我们无法识别5h抗原。少

It is postulated that osteoclast-specific functional proteins are expressed during the process of osteoclastic differentiation. Based on this hypothesis, we attempted the identification of an antigen (5H-antigen) for an osteoclast-specific antibody (Mab-5H) that had been established in our laboratory. Using a experimental system of osteoclastic cell formation in culture of rabbit unfractionated bone cells, we found that the 5H-antigen was expressed in the osteoclast percursors in parallel with the expression of tartrate-resistant acid phosphatase (TRAP) whichis a marker enzyme of osteoclast-diffeerntiation. When Mab-5H simultaneously with parathyroid hormone (PTH) was added into the cultures, the formation and the bone-resorbing activity of osteoclastic cells induced by PTH were abolished by the Mab-5H.These results suggest that the 5H-anitgen specifically produced by osteoclast precursors is involved in the differentiation and function of osteoclasts. In addition, 5H-antigen had two d … More ifferent forms in the osteoclasts, e.g., one was a cytosolic form and the other was a plasma membrane-bound form. Thus, we tried to purify these two distinct forms. However, it was difficult to purify the antigen from osteoclast extracts due to the small number of osteoclasts that we could harvest from the bones. Then, we sought other sources beside osteoclasts to purify the 5H-antigens, and found that intestine also expressed the 5H-antigens enough to purify them. The purified 5H-antigen in a membrane-bound form was a highly-glycosylated protein with molecular mass of 65 kDa in SDS-PAGE analysis, and was very unstable. The cytosolic antigen was also a glycoprotein with molecular mass of 600 kDa under a neutral condition and of 65 kDa in SDS-PAGE,and much more atabel the membrane-bound form. Then we sought the amino acid sequence of the cytosolic 5H-antigen. However, the amino acid analysis was unsuccessful because of a blockage of N-terminus of the antigen. We also tried to analyze the amino acid sequence of peptide fragments of the antigen generated by the limited hydrolysis, but we could not obtain the enough amount of the fragment ot be analyzed. In this study, we found the 5H-antigen was predominantly expressed in osteoclast precursors and playd an important role in osteoclast differentiation and function. However, we could not identify the 5H-antigens. Less